Project Summary The vacuolar H+-ATPase (V-ATPase) is an essential proton pump that is exploited by cancer cells to promote proliferation, migration and drug resistance. In normal cells, this pump creates a defined pH in subcellular organelles that is essential for organelle communication and function, and thus, inextricably ties the V-ATPase to diverse fundamental cellular processes. In normal acid secreting cells, the V-ATPase is found on the plasma membrane where it pumps protons out of the cell, a process required for e.g. urinary acidification and bone resorption. The impact of V-ATPase function on various cellular processes is determined by the membrane on which the enzyme resides, therefore, in normal cells, its abundance and localization are tightly controlled. In many cancers, however, the V-ATPase is upregulated and mislocalized, an essential adaptation for cancer survival. Indeed, inhibition of the V-ATPase leads to suppression of metastasis, increased drug sensitivity and ultimately, cancer cell death. However, total loss of V-ATPase function is embryonic lethal and most of the enzyme's ~15 different subunits are expressed as multiple isoforms, imposing significant barriers to both the study and therapeutic targeting of the enzyme. Importantly, subunit a exists as four isoforms (a1-4), with differential tissue and (sub)cellular localization and recently, specific isoforms have been shown to be overexpressed and mislocalized in breast (a3, a4) and ovarian cancers (a2). Further, our preliminary data indicates that a4 is highly upregulated in renal cancers. The long term objectives of this work are to improve cancer patient outcomes by revealing novel targets for therapeutic development, namely subunit isoforms of the human V-ATPase. The immediate goal of the here proposed work is to generate a powerful new tool, single domain antibodies or Nanobodies (Nbs), for the study of specific V-ATPase populations. Nbs are derived from the unique heavy chain antibodies found in Camelidae and have many advantages over traditional antibodies. For example, Nbs are small, highly stable, and can be used intracellularly. We will use biochemical and biophysical methods, live cell imaging and cell based assays in the following Specific Aims: 1.) Generation and characterization of Nanobodies against subunit a isoforms of human V-ATPase and 2.) Nb mediated characterization and ablation of V-ATPase isoform a4 in kidney cancer. This project is aimed at overcoming the current limitations in the study of V-ATPase isoforms by developing novel and innovative tools, which will have highly transformative potential for the understanding of specific V-ATPase populations in human health and disease. At the end of the proposed research, we expect to have established Nbs as a powerful means to study isoforms of the V-ATPase and that implementation of these Nbs will illuminate the specific role of isoform a4 in promoting kidney cancer survival and malignant phenotypes. The information generated as a result of these studies will provide a firm foundation for developing novel therapeutics for subunit isoform specific targeting of V-ATPase populations in cancer.